Summary: Positive Aspects of Negative Design: Simultaneous Selection of Specificity and
Interaction Stability
Jody M. Mason, Kristian M. Mu¨ller, and Katja M. Arndt*
Institute of Biology III, Albert-Ludwigs UniVersity of Freiburg, Scha¨nzlestrasse 1, D-79104 Freiburg, Germany
ReceiVed December 5, 2006; ReVised Manuscript ReceiVed February 6, 2007
ABSTRACT: The energetic determinants that drive specific protein-protein interactions are not entirely
understood. We describe simultaneous in ViVo selection of specific and stable interactions using homologous
peptides which compete with protein libraries for an interaction with a target molecule. Library members
binding to their target, and promoting cell growth, must outcompete competitor interactions with the
target (i.e., competition) and evade binding to the competitors (i.e., negative design). We term this a
competitiVe and negatiVe design initiatiVe (CANDI). We combined CANDI with a protein-fragment
complementation assay (PCA) and observed major specificity improvements, by driving selection of winning
library members that bind their target with maximum efficacy, ensuring that otherwise energetically
accessible alternatives are inaccessible. CANDI-PCA has been used with libraries targeted at coiled coil
regions of oncogenic AP-1 components cJun and cFos. We demonstrate that comparable hydrophobic
and electrostatic contributions in desired species are compromised in nondesired species when CANDI is
executed, demonstrating that both core and electrostatic residues are required to direct specific interactions.
Major energetic differences (g5.6 kcal/mol) are observed between desired and nondesired interaction
stabilities for a CANDI-PCA derived peptide relative to a conventional PCA derived helix, with
significantly more stability (3.2 kcal/mol) than the wild-type cJun-cFos complex. As a negative control,